Background technology
Soybean protein isolate is to be a kind of protide food additives of raw material production with the big dregs of beans of low temperature desolventizing, nitrogen content is more than 90% and have multiple functional characteristic such as emulsibility, gelling, water proofing property etc., its nutritious cholesterol that do not contain, be one of kind of alternative animal protein few in number in the vegetable protein, nutritive value can match in excellence or beauty with beef, egg, milk etc.
Soybean protein isolate viscosity significantly increased with the increase of concentration, just began to form gel usually at 12% o'clock.This point has limited protein isolate greatly in Application in Food.The albumen that requires to add wherein such as some baby food and dairy products can not harden or precipitate owing to the prolongation of product standing time, beverage with reconstitute beverage and can not have gelation with protein isolate.For fear of the gel of soybean protein isolate may, soybean protein isolate can carry out enzyme hydrolysis earlier before being applied to this series products.
As far back as the seventies and eighties in 20th century, U.S. Adler-Nissen etc. has just made number of research projects to the enzyme hydrolysis of soybean protein, think that enzyme hydrolysis can destroy the gelation of soybean protein, but, normally be difficult to the albumen of the such native form of this hydrolase decomposing soya-bean protein of protease.Main component in the soybean protein isolate is 7s and 11s albumen.Polypeptide chain closely folds, and hydrophobic amino acid portion within it forms hydrophobic region, and the outside is wrapped up by hydrophilic outer shell, and these subunits are bonded to each other again and form complicated spherical quaternary structure, so the soybean protein molecule has very strong resistance to enzymolysis.If wish to get the protein isolate series products, can adopt the method for heating that protein part is launched earlier, hydrophobic grouping is partly exposed, promptly physical modification is hydrolyzed again to albumen earlier.
About the sex change and the hydrolysis mechanism of globulin,
![Figure G2007100212575D00011](https://patentimages.storage.***apis.com/79/8e/46/ece2a91d17071a/G2007100212575D00011.png)
Proposed such notion in nineteen fifty-two: native protein is earlier through sex change, obtain the intermediate state product of an allosteric through first one-step hydrolysis again, thus then enzyme further effect obtain final products.If sex change speed is much smaller than first step hydrolysis rate, hydrolysis is controlled by sex change speed, reactant mixture has only native protein and end-product, and this reaction is called as " (the one by one) reaction of going forward one by one ", and this reaction means a kind of substrate of the once single-minded degraded of a kind of protease.On the contrary, if first step hydrolysis rate is much smaller than sex change speed, native protein molecule fast transition is an intermediate state, the intermediate state product slowly is degraded to end-product again, Here it is " zippering reaction (zipper reaction) ", that is to say when sex change speed is big, first step hydrolysis rate just become have decisive, at least in the reaction starting stage, overall reaction rate will improve.When low in hydrolysis was spent, the soluble part of the reaction of going forward one by one comprised native protein and few small peptide.And if chain reaction, solubilized partly is exactly the peptide section of molecular weight distribution broad.And if the native form of protein substrate is followed the reaction rule that goes forward one by one, the result of sex change can improve reaction rate exactly, and the speed of degraded is similar to zippering reaction.
Adler-Nissen once did experimental study, the control degree of hydrolysis is 6%, defatted soy flour directly through Alcalase hydrolysis and defatted soy flour through pH4.5 following 50 ℃ of heating 10 or 30 minutes again or 90 ℃ of heating hydrolysis is relatively again after 10 minutes, product detect to be found by gel chromatography: the hydrolysate of natural substrate is mainly albumen or big peptide chain and rare little peptide that can not be separated, and the hydrolysate of sex change substrate is the peptide section of moderate-length of relative broad range and the peptide section of rare macromolecule.Research afterwards shows that further the dissolubility of the unmodified relatively albumen of the peptide chain of moderate-length in acid solution will get well, and this protein product is commonly used in dairy products and the acidic beverages.
Folding energy barrier theory (energy landscape theory) and the leaky bucket concept (the funnel concept) of modern albumen (going) pointed out, is not a step to finish unfolding and become complete deployed condition during protein denaturation.It between the different proteins structure relation a kind of rigidity, either-or; From native state to the sex change attitude, there is the transition state that is referred to as molten spheroid, the degenerative process that is albumen is: the molten ball transition state that is changed into stable momentary partial sex change by the natural folding attitude, change complete deployed condition again into, the natural structure in the amino residue just might become the non-natural attitude in processing procedure.People such as P.X.Qi and E.M.Brown has further studied the structural change of lactoprotein, proves the process from native state to the sex change attitude also to have multiple intermediate state.Research has afterwards proved that further the breakdown rate of globular preteins peptide bond greatly is subjected to the influence of tertiary structure.Because the transition state protein molecule has more active group and avtive spot, makes us more effectively to carry out structural modification to it.The denaturation degrees of protein can be controlled by multiple modes such as temperature, pH, heat time heating time, protein concentration, organic solvent and mechanisms.Such as people such as Z.Y.Ju the gelation of enzymatic lactalbumin is studied, they descend heating after 2 to 30 minutes at 80 ℃ 9% lactoalbumin soln, act on bacillus licheniformis (Bacillus licheniformis protease), survey its gelation, find that gel process and denaturation degrees are closely related, denaturation degrees is high more, and gel more early, it is faster that gel hardness increases, and denaturation degrees and gel hardness are linear correlation.Sur ó wka also mentions a kind of method for preparing modified protein earlier through the extruding sex change in his article, by SDS-PAGE relatively Alcalase and Esperase protease to the product of extruding and non-extruding FSPC restricted hydrolysis, find to reach under the situation of same amount ammonia nitrogen with extruding albumen, the hydrolysis of non-extruding albumen has more selectivity, many typical soybean protein chemical bonds still exist, and the raw material ratio after the extruding do not push before facile hydrolysis more.Can see that by above-mentioned theory and example through the soybean protein after the sex change be a kind of brand-new protein isolate.So the condition that heats among the present invention comprises that the preparation of transition state soybean protein and the enzyme that goes out of hydrolyzate all are the key issues that is worth research.From the data of grasping, also there is not the report of this respect both at home and abroad.
Summary of the invention
Purpose of the present invention is exactly to develop a kind ofly partial heat denaturation albumen is carried out enzyme modification and to obtain the soybean protein of gel-free.Confirm that after deliberation after the heat denatured under the isoelectric point soybean protein process controlled condition, by the Restriction Enzyme hydrolysis, its gelation is destroyed.The bitter taste that produces when eliminating soybean protein hydrolysis by the exopeptidase that adds doses makes hydrolysate can be applied to dairy products, beverage and reconstitutes in beverage, proteinaceous nutrient replenishers and the injection-type.
For effectively destroying gelation, adopt the mode of heating albumen under isoelectric pH value, obtain a kind of soybean protein raw material of transition state, the condition of heating is one of main research object among the present invention.
Technical scheme of the present invention: a kind of enzyme modification by the transition state soybean protein prepares the method for low gel property high dispersancy soy protein, and described method comprises:
(1) with the defatted soybean meal is the soybean protein of feedstock production partial heat denaturation, i.e. transition state soybean protein;
(2) enzymolysis of transition state soybean protein: add enzyme preparation successively, with transition state soybean protein hydrolysis certain hour, and in this process debitterize, thereby produce a kind of soybean protein hydrolyate;
(3) post processing: soybean protein hydrolyate inactivator mixture obtains soy protein products through homogeneous, spray-drying, spraying phosphatide.Require the gel strength of product: the gel strength of soft gel state product is about 50g, and the gel strength of gel-free attitude product is less than 25g.
Preparation transition state soybean protein: with the defatted soybean meal is raw material, adopts conventional method to be suspended in solid-to-liquid ratio 1: 10~1: 20g/ml in 30 ℃ the aqueous solution, is molten 40~60 minutes of 7.0~8.0 times alkali in the pH value, and centrifugation obtains the albumen supernatant; It was 4.0~5.0 that supernatant is regulated the pH value, 40~80 ℃ of following agitating heating 5~30 minutes; After the heating of acidic protein solution finished, centrifugation obtained skyr, and adding water to solid content is 8%~18%, and pulling back to the pH value is 7.25~7.4, obtains the transition state soy bean proteinous soln.
The optimum condition of preparation transition state soybean protein is: be raw material with the defatted soybean meal, adopt conventional method to be suspended in solid-to-liquid ratio 1: 10~1: 20g/ml in 30 ℃ the aqueous solution, in the pH value is molten 40~60 minutes of 7.0~7.5 times alkali, and centrifugation obtains the albumen supernatant; It was 4.0~5.0 that supernatant is regulated the pH value, 50~60 ℃ of following agitating heating 5~10 minutes; After the heating of acidic protein solution finished, centrifugation obtained skyr, and adding water to solid content is 10%~14%, and pulling back to the pH value is 7.25~7.4, promptly obtains the transition state soy bean proteinous soln.
The enzymolysis of transition state soybean protein: in the transition state soy bean proteinous soln that obtains, add 0.1%~0.5% endopeptidase and 0.05%~0.5% exopeptidase successively in the soybean protein solid content, 50~60 ℃ of hydrolysis temperatures, enzymolysis time 30~40min;
Described endopeptidase is the mixture of neutral bacterialprotease, plant rennet or two kinds of enzymes;
Described exopeptidase is Mycophyta protease: Flavourzyme (Novo) and Peptidase R (Amano).
The enzymolysis liquid post processing: the hydrolyzate that obtains adopts and is steam heated to 100~140 ℃, effect 5~10s enzyme that goes out; The homogeneous behind the enzyme that goes out, homogenization pressure is 25~35MPa; Spray-dired EAT is 160~180 ℃, and leaving air temp is 60~85 ℃; Phosphatide emulsion with 20% is sprayed at the albumen powder surface with 1: 10 ratio, promptly gets product.
The optimum condition of enzymolysis liquid post processing: the hydrolyzate that obtains adopts and is steam heated to 120~140 ℃, effect 5~10s enzyme that goes out.
Dregs of beans is through the molten centrifugal protein solution that obtains of alkali, regulates 40~80 ℃ of heating after the isoelectric point scope of pH value, obtains acid transition state protein raw materials after the centrifugation.The temperature and time of heating should be noted that.Because the difference of treatment conditions can produce different transition state albumen, thereby influence the functional of the condition of hydrolysis and product.Go back to adjust pH to 7.25~7.4, controlling such initial pH value is in order no longer to regulate the Acidity of Aikalinity of protein solution after finishing in reaction.Along with pH is close to neutrality, viscosity raises suddenly, and pH transfers to ideal range and becomes difficult.Therefore, when in skyr, adding alkali, must constantly stir.And for preventing excessive alkali zone and the heat release point of generation in the skyr, the dilution of alkali lye also is necessary, otherwise can cause some amino acid whose decomposition.Add 0.1%~0.5% endopeptidase and 0.05%~0.5% exopeptidase in the soybean protein solid content, 50~60 ℃ of effect 30~40min.Behind the hydrolyzate homogeneous that obtains, adopt to be steam heated to 100~140 ℃, effect 5~10s enzyme that goes out finishes back homogeneous, spray-drying, spraying phosphatide and obtains soy protein products.
It is as follows that the product that obtains is surveyed its every performance methodology:
(1) gelation: get the 3g soybean protein it is dissolved in the 22g deionized water, promptly be made into 12% protein solution in the 25ml beaker, seal with preservative film, in 90 ℃ of water-baths, take out behind the heat tracing 30min, place ice-water bath to cool off rapidly, put into and observe after 4 ℃ of refrigerators are preserved 24h, measure gel strength with property tester (TextureAnalyzer TA-XTZi), selecting diameter for use is the cylindric tack drift of 12mm, drawing velocity 4mm/s, required power was defined as gel strength when the record gel broke, and required power was gel elastomer when the record press depth was 5mm.
(2) in the TCA-NSI trichloroacetic acid soluble nitrogen assay (A 5% TCA Solubilization Byweight) but---the hydrolysis situation of the method profiling protein.Get the 1g modified soy-bean protein and be dissolved in the 24g deionized water, the trichloroacetic acid solution of quality 10% such as adding, fully vibration mixes, leave standstill 10min, centrifugal 20min under the 4000r/min rotating speed gets the certain proportion supernatant legal nitrogen of kjeldahl determination, 5%TCA-N dissolubility (wt%)=N
1/ N
0* extension rate, (N in the formula
1Be soluble nitrogen in 10% trichloroacetic acid, N
0Be total nitrogen in the soybean protein).
(3) nephelometry is measured suspension stability---the soy bean proteinous soln of preparation 0.4%, absorbance intensity of variation (the Δ ε %=(ε of its 24 hours front and back of observation under 600nm
0-ε)/ε
0* 100%), to characterize the suspension stability of solution.
Beneficial effect of the present invention: the present invention finds can to obtain after thermal denaturation transition state soybean protein is by enzyme modification the soybean protein a little less than the gelation.And the suitable enzyme processing of going out then can take into account the suspension stability that the destruction gelation does not influence protein solution.
The suspension stability of our denaturation degrees by discovering albumen and its protein hydrolysate exists the relation that conditions each other.Heating intensity is big more, and it is high more that albumen launches degree, helps enzyme hydrolysis more and destroys gelation, there are some researches show that the viscosity of protein solution of moderate-length peptide chain of the molecular weight of consisting of 5,000 to 20,000 is lower; But excessive heating then can cause the gathering of protein hydrolysate peptide section, thereby influences the suspension stability of solution.This shows that it is significant how to control denaturation degrees before the enzyme modification.
Except good suspension stability, the soybean protein that is used for food also must have good local flavor.Generally, for the hydrolysis of albuminate, the hydrolysate of moderate-length peptide chain is main, but the small peptide that meanwhile produces has caused offending bitter taste.Thereby the adding with Mycophyta protease of exopeptidase activity can be played the purpose that the excision hydrophobic grouping reaches debitterize.
Our national dairy products and the injection of piece meat products are produced desirable proteins and are all relied on from external import always.The reason that forms this situation mainly is that the key production technology thereof technology does not have breakthrough, and some advanced modified techniques also are not applied in the production.So the present invention has good promotion prospect.
The specific embodiment
Embodiment 1
With the defatted soybean meal is raw material, adopt conventional method to be suspended in solid-to-liquid ratio 1: 15g/ml in 30 ℃ the aqueous solution, in the pH value is the molten 50min of 7.4 alkali, centrifugation obtains supernatant, after supernatant adjusting pH value is 7, and 50 ℃ of agitating heating 10min, centrifugation obtains skyr, adding water to solid content is 13%, and returning adjust pH is 7.4, adds 50~60 ℃ of effects of 0.2% hydrolysising protease and 0.3%Peptidase R 40min.The hydrolyzate that obtains adopts and is steam heated to 120 ℃ of effect 5s enzyme that goes out, and finishes the back homogeneous, obtains soy protein products behind spray-dried, the spraying phosphatide.The product gel strength is 102.3g, and gel elastomer is 76.7g, and 5%TCA-N dissolubility (wt%) is 13.4%, and Δ ε % is 3.5%.
Other conditions are in the listed condition and range of the present invention, and only supernatant accent pH is 7.0 o'clock, and the gel strength of product is tested undesirable greater than 100g as a result.
Embodiment 2
With the defatted soybean meal is raw material, adopt conventional method to be suspended in solid-to-liquid ratio 1: 15g/ml in 30 ℃ the aqueous solution, in the pH value is the molten 50min of 7.4 alkali, centrifugation obtains supernatant, after supernatant adjusting pH value is 4.5, and 80 ℃ of agitating heating 5min, centrifugation obtains skyr, adding water to solid content is 13%, and returning adjust pH is 7.25, adds 50~60 ℃ of effects of 0.1% endopeptidase and 0.05%Peptidase R 40min.The hydrolyzate that obtains adopts and is steam heated to 120 ℃ of effect 5s enzyme that goes out, and finishes after obtain soy protein products behind the homogeneous, spray-drying, spraying phosphatide.The product gel strength is 4.9g, and gel elastomer is 3.1g, and 5%TCA-N dissolubility (wt%) is 67.5%, and Δ ε % is 73.5%.
Embodiment 3
With the defatted soybean meal is raw material, adopt conventional method to be suspended in solid-to-liquid ratio 1: 10g/ml in 30 ℃ the aqueous solution, in the pH value is the molten 60min of 7.2 alkali, and centrifugation obtains supernatant, after supernatant adjusting pH value is 4.5,40 ℃ of agitating heating 30min, centrifugation obtains skyr, and adding water to solid content is 14%, and returning adjust pH is 7.3, add 0.5% endopeptidase and 0.10%Peptidase R, 50~60 ℃ action time 40min.The hydrolyzate that obtains adopts and is steam heated to 120 ℃ of effect 10s enzyme that goes out, and finishes after obtain soy protein products behind the homogeneous, spray-drying, spraying phosphatide.The product gel strength is 53.2g, and gel elastomer is
Fig. 1
Fig. 2